Changes in Intestinal Fluid Transport and Immune Responses to

Vol. 30, No. 3

INFECTION AND IMMUNITY, Dec. 1980, p. 734-740 0019-9567/80/12-0734/07$02.00/0

Changes in Intestinal Fluid Transport and Immune Responses to Enterotoxins Due to Concomitant Parasitic Infection INGER LJUNGSTROM,`* JAN HOLMGREN,2 GUNNEL HULDT,' STEFAN LANGE,2 AND ANN MARI SVENNERHOLM2 Department of Parasitology, National Bacteriological Laboratory, S-105 21 Stockholm,' and Institute of Medical Microbiology, University of Goteborg, S-413 45 Goteborg,2 Sweden

The effect of a parasitic infection on enterotoxic diarrhea and on local and systemic formation of antibody to the toxin after immunization was studied in mice. Trichinella spiralis infection was chosen as the model, since the effects of the parasite when residing in both intestinal and extraintestinal sites can be studied. It was found that during the intestinal stage of the infection, the fluid response to cholera toxin as well as dibutyryl-cyclic adenosine 3',5'-monophosphate was greatly enhanced and that this was associated with a marked reduction in the absorption of fluid from the intestine. Later in the infection (migration stage), fluid accumulation in response to cholera toxin was significantly reduced, whereas absorption was normal and secretion in response to dibutyryl-cyclic adenosine 3',5'-monophosphate was somewhat increased. Still later in the infection (muscular stage), the fluid-secretory response to cholera toxin was normal. There was a drastic depression of local formation of antitoxin of both immunoglobulin and immunoglobulin classes in mice given the first two of four oral immunizations with cholera toxin during the intestinal stage of T. spiralis infection. When the priming was given before or after the intestinal stage, the local antitoxin response was not affected. The titers of circulating antibodies were also depressed in mice given the first immunizations during the intestinal stage. In addition, significant though less pronounced depression of the serum antibody response was observed in mice primed during the extraintestinal stage. Acute diarrhea is a worldwide health problem, effects of parasites on the local immune rebut despite this, very little is known about the sponses to unrelated antigens, such as enterotoxgenetic and environmental factors governing the ins, are largely unknown. Such effects could individual responses to enteric pathogens. obviously be important for the susceptibility to Among environmental factors, the intestinal mi- and the outcome of enteric infections as well as croecology is an important but unexplored area. for responses to vaccination against such disIn populations with poor hygienic conditions, eases. intestinal parasites are common. The effects of In the present study we have explored the these often abundant organisms on the intestinal possibility that an intestinal parasitic infection ecology and responses to other enteric pathogens could be an environmental factor modulating are largely unknown. the host response to enterotoxin-producing bacEnterotoxin-producing bacteria are responsi- teria. The effects of trichinosis on experimental ble for a high proportion of acute diarrheal dis- cholera and cholera immunization in mice were eases. It is conceivable that intestinal parasites investigated. The specific aims were: (i) to deresiding in the unstirred layer of mucus in close termine whether trichinosis in its intestinal and contact with the epithelial cells or in the lamina extraintestinal stages would affect fluid transpropria could interfere with the effects of bac- port processes, resulting in a modified diarrheal terial enterotoxins on intestinal fluid transport response to cholera toxin, and (ii) to study the mechanisms. Several parasites are also known possibility that the parasitic infection might to have serious suppressive effects on immune modulate the local immune response in the gut responses (19). Although the majority of these to cholera toxin immunization. parasites have a systemic localization, recently MATERUILS AND METHODS also some intestinal parasites have been found to depress various immune responses. Among Cholera toxin. The cholera toxin used was a highly these, Trichinella spiralis is the one most ex- purified preparation purchased from Schwarz/Mann, tensively studied (16). However, the possible Orangeburg, N.Y. 734

VOL. 30, 1980

T. SPIRALIS EFFECT ON RESPONSE TO ENTEROTOXINS

Mice. Inbred C57BL/6J (H-2b) mice of both sexes were used. The animals were closely age and sex matched within each experiment. The age varied between 5 and 7 weeks at the onset of infection or immunization. Parasitic infection. The strain of T. spiralis used in these experiments has been maintained at the National Bacteriological Laboratory, Stockholm, Sweden, for more than 30 years by passage through Sprague-Dawley rats. Larvae were recovered from infected rat muscles by HCl-pepsin digestion (14) and used for the experimental infections. Each mouse was inoculated with 500 larvae through a stomach catheter. In the small-intestinal mucosa the larvae rapidly develop into adult worms, which by 5 days start to produce larvae (intestinal stage). About 2 weeks after infection the vast majority of the adult worms are expelled from the gut, but there is a large population of new larvae migrating through the vascular system to the muscles (migration stage). After 3 weeks no parasites are found in the intestine; instead, the infection proceeds in striated muscles (muscular stage). Assays of intestinal secretion and absorption. Intestinal fluid secretion in response to cholera toxin was studied in ligated small-bowel loops (11). Two 5to 7-cm-long jejunal loops were prepared, one of which was inoculated with 0.2 ml of cholera toxin in phosphate-buffered saline (PBS) (0.05 M phosphate, 0.14 M NaCl, pH 7.2) and the other of which was inoculated with 0.2 ml of PBS only. After 4 to 5 h (when not stated otherwise) the animal was killed, and the length and weight of the loops were determined. Fluid accumulation attributable to the toxin was estimated as the weight difference (milligrams per centimeter) between the test and control loops. The intestinal fluid response to dibutyryl-cyclic adenosine 3',5'-monophosphate (dBcAMP; Sigma Chemical Co., St. Louis, Mo.) was also tested (8). A single jejunal loop was arranged into which 1.5 mg of dBcAMP dissolved in 0.2 ml of PBS was injected. Fluid accumulation was determined after 90 min. Intestinal fluid absorption was studied in ligated jejunal loops. The loops were injected with 0.2 ml of Ringer solution, and the fluid content was measured immediately and after 15, 30, or 60 min (8). Immunizations. Groups of T. spiralis-infected and uninfected mice were immunized with cholera toxin. Four immunizations, each consisting of 5 tg of cholera toxin in 0.5 ml of 5% sodium bicarbonate, were given perorally by a stomach tube. The interval between the first and second immunization was 7 days, and subsequent immunizations were given at 6-day intervals. Immunity was evaluated 4 days after the last immu-

nization. Enzyme-linked immunosorbent assay of serum antibody. Antibodies of the immunoglobulin G (IgG) and IgA classes in serum were determined by means of the enzyme-linked immunosorbent assay, using purified cholera toxin as the solid-phase antigen (17). The antibody titer was defined as the extrapolated dilution of serum giving rise to an absorbance change of 0.4 above background in 100 min with the anti-IgA or anti-IgG enzyme conjugate. IgM antibodies were not determined, since our previous work has shown that these antibodies are formed in very low

735

amounts after oral immunization and have no protec-

tive significance (17). Intestinal antibody synthesis. Antibody synthesis by in vitro-cultured intestinal tissue was determined as described previously (17). Briefly, the small intestine was quickly excised, and multiple tissue specimens were taken from different parts. Equal amounts (approximately 500 mg) of tissue were taken from experimental animals and controls. The tissue was thoroughly washed and minced into small pieces. The tissue was then incubated at 370C for 24 h in Eagle medium supplemented with 5% heat-inactivated normal serum and 200 U of penicillin-streptomycin per ml at a constant pH of 7.2 to 7.4. After incubation the medium was centrifuged at 3,000 x g for 10 min, and the supernatant was recovered and frozen at -30'C. The titers of specific IgA and IgG antibodies to cholera toxin were determined with the enzyme-linked immunosorbent assay. ACC. Specific cholera antitoxin-containing cells (ACC) in intestinal laminae propriae were determined by a peroxidase labeling procedure (10). Five-millimeter-long intestinal specimens from the mid jejunum were fixed in ice-cold formaldehyde-picric acid, washed in PBS, dehydrated in ethanol, embedded in paraffin, and cut into 5-Mm-thick transverse sections. After deparaffinization and incubation with Triton X100 supplemented with 5% bovine serum albumin, the specimens were treated with 1 IMM 1,4'-aminotriazole in PBS to eliminate endogenous peroxidase activity. The specimens were then washed and incubated with cholera toxin (1 ug/ml in PBS) for 90 min at room temperature. After another washing with PBS, the specimens were incubated with peroxidase-conjugated rabbit anti-cholera toxin (10). The sections were incubated for 5 to 10 min at room temperature with 3',3'-diaminobenzidine (0.5 mg/ml, Sigma) containing 0.01% hydrogen peroxidase (Perhydrol; E. Merck AG, Darmstadt, Germany). After rinsing in PBS, the sections were dehydrated in ethanol and then embedded for microscopic examination. The number of ACC was determined on 10 transverse intestinal sections from 4 to 6 mice in each experiment; from these values the number of ACC per cubic millimeter of lamina propria was calculated as described previously (12). RESULTS

Cholera toxin-induced fluid secretion. Intestinal fluid secretion in response to cholera toxin was determined in ligated loops at various times after T. spiralis infection. Marked differences in the secretary response at the various stages of the infection were observed (Fig. 1). When the parasite resided mainly in the intestine (day 7), the fluid accumulation was significantly increased in comparison with that in concurrently tested uninfected mice. In contrast, later in infection, when the majority of the adult worms were expelled and there was an ongoing migration of newborn larvae to the muscles, the fluid-secretory response was markedly decreased. At a further, later stage (day 41), when the infection was confined to the striated mus-

736 LJUNGSTROM ET AL.

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INFECT. IMMUN.

Intestinal cholera antibody synthesis. The possibility that the parasitic infection might affect the ability of the intestine to respond immunologically to cholera toxin antigen was tested. The mice were given four peroral im-

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cles, the fluid accumulation in response to cholera toxin was the same as in uninfected mice (Fig. 1). The kinetics of fluid secretion after challenge with cholera toxin was also tested. The experiments showed that there was no difference in the time course of fluid accumulation between parasite-infected and uninfected animals. The maximal response was recorded 5 h after the exposure to toxin and proportionally differed more after a low than after a high toxin dose (Fig. 2). Intestinal fluid accumulation reflects secretory as well as absorptive processes. The influence of the parasitic infection on intestinal fluid absorption was tested in ligated jejunal loops. The results showed that the net absorption of isotonic Ringer solution was profoundly decreased during the intestinal stage of infection, whereas it had almost fully recovered at the time for or after the migration of the parasites from the intestine (Fig. 3). Fluid accumulation in response to dBcAMP was compared with that to cholera toxin in order to test whether the alterations caused by T. spiralis infection were similar for preformed cyclic AMP and the adenylate cyclase-activating enterotoxin. During the intestinal stage the increase in fluid accumulation was greater in response to dBcAMP than to cholera toxin (Fig. 4). Later in the infection, when a decreased response to cholera toxin was seen, the response to dBcAMP was still slightly elevated in comparison with the uninfected controls.

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FIG. 2. Time course of fluid accumulation in response to 1.5 pg (open symbols) and 6 fig (closed symbols) of cholera toxin measured in ligated jejunal loops in T. spiralis-infected (solid lines) and uninfected (broken lines) mice. Mean ± standard error of the mean (S.E.M.) of results from five mice is shown.

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VOL. 30, 1980

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FIG. 4. Comparison of intestinal fluid secretions in response to dBcAMP (0) and cholera toxin (X) at various times after T. spiralis infection. The values express the percent fluid accumulation (± standard error of the mean [S.E.M.]) in infected animals compared with concurrently tested uninfected animals. The dBcAMP challenge was 1.5 mg per loop, which gave a fluid accumulation of 41 ± 7 mg/cm in the uninfected animals, and the cholera toxin challenge was 2.5 jig per loop (145 ± 11 mg/cm). The challenge doses used were approximately half of the doses giving maximal effect.

737

extraintestinal stage, starting on day 18 (Fig. 5D). Antitoxin-containing cells in lamina propria. The effect of T. spiralis infection on the local immune response to cholera toxin immunization was also studied in terms of the number of ACC in the intestinal lamina propria. In accordance with the in vitro antibody synthesis, the ACC response was profoundly depressed in animals immunized twice during the intestinal stage and twice during the extraintestinal stage (Table 1). No significant effects on the ACC response were seen when immunizations started before the T. spiralis infection and only the very last or the two last immunizations were given after infection during the intestinal stage (Table 1). Serum antibodies. The IgG and IgA antibody responses in serum were also studied (Fig. 6). Both IgG and IgA titers were markedly depressed when the immunization started on day 5 (Fig. 60). The infection had much less effect on the serum antibody response when two immunizations were given before the infection, starting on day 8 before (Fig. 6B). Thus, the effects of T. spiralis infection on the serum antibody titers corresponded to the effects on the local immune response. However, in T. spiralis-infected mice in which the whole immunization schedule was carried out in the extraintestinal stage, starting on day 18, there was a greater depression of serum antibodies of both the IgA and IgG classes (Fig. 6D) than of intestinally synthesized antibodies.

munizations with cholera toxin according to a schedule known to induce optimal immune pro103r tection against experimental cholera (8). The mucosal immune response was determined as A B the amount of antitoxin antibodies in various immunoglobulin classes synthesized in vitro by 4 the tissue-cultured intestine. In uninfected mice . ,. i,. 102 the immunization gave rise to significant local production of mainly IgA antibodies but also cr44 some IgG antitoxin (Fig. 5A). No detectable Io_P synthesis of antibody to cholera toxin was ob1 served in nonimmunized animals, irrespective of z II 101 the mice were infected In whether (not shown). T. spiralis-infected mice given two immuniza- Ls 101 tions during the intestinal stage, starting on day 18 CONTROL -8 5 START OF IMMUNIZATION(day) IN 5 after infection, and the other two during the TO T.spiralis INFECTION RELATION extraintestinal stage, there was a very marked FIG. 5. Effect of T. spiralis infection on in vitro decrease in the local formation of both IgA and IgG antibodies (Fig. 50). T. spiralis infection antibody synthesis in the small intestine after oral had much less effect on the local antibody re- immunization with cholera toxin. For the immunization schedule, see the test. Shaded bars represent sponse when two immunizations were given be- IgA and open bars represent IgG antibodfore the infection, starting on day 8 before, and ies. antibodies, The values shown are the means (± standard the last two immunizations were given in the error of the mean [S.E.M.J) of the results from two intestinal stage (Fig. 5B) or when the whole experiments in which antibodies were determined in immunization scheme was carried out in the triplicate on pooled specimens from three animals.

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738 LJUNGSTROM ET AL.

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TABLE 1. Effect of T. spiralis on the number of ACC in intestinal lamina propria after peroral immunization with cholera toxin' No. of immunizations, stage

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errr) 4, Uninfected 2,647 ± 204 28 447 ± 78 83 0.10 intestinal 3, Uninfected; 1, 7 2,563 ± 166 3 >0.10 intestinal " Four immunizations were given with 5 ug of cholera toxin. The interval between the first and second immunization was 7 days, and subsequent immunizations were given at 6-day intervals. The number of ACC was tested 4 days after the last immunization. Inhibition was calculated in relation to results with uninfected mice.

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FIG. 6. Serum antibody responses to cholera toxin in T. spiralis-infected and uninfected mice. The results are expressed as the means (± standard error of the mean [S.E.M.]) of repeated titrations ofpooled specimens and were obtained on sera from the same groups of animals that were investigated for intestinal antibody synthesis (see Fig. 5). Shaded bars are IgA antibody titers, and open bars are IgG antibody titers.

DISCUSSION The effects of an intestinal parasitic infection on enterotoxic diarrhea and on the local and systemic immune response to the toxin were studied in a mouse model system. Cholera toxin was chosen because it is the best characterized enterotoxin and since methods recently have been developed to measure local antibody response to this antigen (10, 17). The parasite used in the studies was T. spiralis. Since this parasite spends only part of its life cycle in the small

intestine and the remaining part in extraintestinal sites, it offers unique possibilities to study the effects of the same parasite in different locations. The results show that infection with T. spiralis has a profound effect on intestinal secretion as well as on both local immunity and systemic immunity to cholera toxin. It was found that during the intestinal stage of infection, fluid accumulation in response to cholera toxin was greatly enhanced. The reason for this is probably complex. Intestinal fluid accumulation is the net result of absorption and secretion by both passive diffusion and active transport mechanisms (6). Our results, in agreement with findings by Castro et al. (4), indicate that the main effects on fluid transport exerted by the parasite in its intestinal location are associated with absorption. Several mechanisms might be responsible for the observed inhibition of intestinal fluid absorption in trichinosis. (i) Increased hydrostatic tissue pressure due to inflammatory mucosal edema could be the cause. (ii) Increased turnover of epithelial cells could also be responsible. Proliferation of the intestinal epithelium mainly takes place in the crypts, and the new cells are continuously moved upwards. It has been found that active absorption is mainly effectuated by mature villus cells, whereas cells in the crypts instead mediate active electrolyte and water secretion (6). An increased turnover could thus result in a greater proportion of functionally immature cells on the villus tips with reduced absorptive capacity. There is evidence suggesting that the gastrointestinal hormone gastrin might stimulate epithelial cell turnover and' thereby decrease intestinal absorption (9, 13). It has been demonstrated that T. spiralis causes an increase in serum gastrin (1), and a significance increase in cell proliferation in the intestinal crypts has been observed in rats infected with the related parasite Nippostrongylus brasiliensis (18). (iii) Another possible cause is stimulation of prostaglandin products from inflammatory cells present in the lamina propria in acute intestinal trichinosis. Prostaglandins can increase the cyclic AMP content in the intestinal epithelium and thereby inhibit active absorption as well as stimulate active secretion in essentially the same manner as cholera toxin (6). (iv) Release from the parasite of biologically active substances which might interfere directly with intestinal ion transport mechanisms could also be responsible. It has been demonstrated that T. spiralis releases a number of amines (3, 7), and it is conceivable that some of these could have such effects. An interesting observation is that later in in-

VOL. 30, 1980


fection, when the majority of the parasites had left the intestine (migrations stage), the fluid accumulation in response to cholera toxin was markedly reduced. Our results indicate that this was not due to changed absorption capacity, since absorption of Ringer solution by T. spiralis-infected mice did not differ significantly from that by uninfected control animals. Therefore, it seems more likely that the secretary process was affected. The experiment with dBcAMP did not suggest any inhibition in fluid secretion in response to preformed cyclic AMP. It might therefore be the synthesis of the nucleotide rather than subsequent events in the fluid secretion chain that was impaired in the fluid response to cholera toxin. A subnormal adenylate cyclase activity in the T. spiralis-infected mice already in the intestinal stage of infection might also explain why the fluid response enhancement in infected compared with normal animals was less pronounced in response to cholera toxin than to dBcAMP. The local antibody response to oral immunization with cholera toxin was studied with two independent methods. Both gave comparable results. When immunization was started during the intestinal stage, an almost complete abolishment of intestinal synthesis of both IgA and IgG antibodies was recorded associated with a marked reduction in the number of ACC in the lamina propria. When priming was performed before infection and immunization was continued during the intestinal stage or when all immunizations were given during the extraintestinal stage of infection, there was no significant difference in local antibody response as compared with the uninfected controls. It has to be considered that during intestinal trichinosis the absorption capacity of the enteric epithelium is decreased and simultaneously the intestinal motility is increased (2). It therefore seems conceivable that the amount of antigen reaching immunocompetent cells may have been lower in T. spiralis-infected mice than in controls. However, also when immunization was started on day 8 before infection and two antigen doses were given before and two during the intestinal stage, no decrease of local or systemic antibody response compared with controls was recorded. Earlier studies have shown that the antitoxin response in the intestines of mice is transient, reaching its maximum after 4 days and then declining within 7 to 10 days; however, concomitantly, immunological memory of a longer duration also develops which is boosted by renewed immunizations (11). Our present data indicate that the depressive influence of trichinosis on mucosal antitoxin immunity is confined to the

739

intestinal stage of infection. They also suggest that the depression essentially affects the buildup of immunological memory for the local antibody response rather than the actual booster response. Earlier studies have shown that during intestinal trichinosis, T-cell reactivity is significantly reduced (15). The observed inhibition of local antitoxin production is in agreement with these findings, and it is conceivable that the decreased antibody response was due to T suppressor-helper cell imbalance. Further studies are in progress to elucidate this problem. Possibly, metabolic substances from T. spiralis are the stimulatory or suppressive factors involved. In addition, the levels of circulating IgG and IgA antibodies were significantly decreased in mice given the first immunization during the intestinal stage. There is reason to assume that this decrease was due to the combined effect of local and systemic immune depression. It has been shown that already at 7 days after infection, spleen T cells have a decreased reactivity (15) reflecting an early systemic immune depression. When the oral immunization was started during the extraintestinal stages of the T. spiralis infection, the serum antibody response still was depressed, although to a lesser extent. Since this immunization schedule did not result in depression of local antibodies, it is likely that the decreased response with regard to serum antibodies was due entirely to systemic immune depression. ACKNOWLEDGMENT Hikan Nygren is gratefully acknowledged for the preparation of the peroxidase conjugate. LITERATURE CITED 1. Castro, G. A. 1976. Spatial and temporal integration of host responses to intestinal stage of Trichinella spiralis: retro- and prospective views, p. 343-358. In H. van den Bossche (ed.), Biochemistry of parasites and hostparasite relationship. Elsevier/North-Holland Biochemical Press, Amsterdam. 2. Castro, G. A., F. Badial-Aceves, J. W. Smith, S. J. Dudrick, and N. W. Weisbrodt. 1976. Altered small bowel propulsion associated with parasitism. Gastroenterology 71:620-625. 3. Castro, G. A., J. D. Fergusson, and C. W. Gordon. 1973. Amine excretion in excysted larvae and adults of Trichinella spiralis. Comp. Biochem. Physiol. 45A: 819-828. 4. Castro, G. A., J. J. Hessel, and G. Whalen. 1979. Altered intestinal fluid movement in response to Trichinella spiralis in immunized rats. Parasite Immunol. 1:259-266. 5. Cebra, J. J., R. Kamat, P. Gearhart, S. M. Robertsson, and J. Tseng. 1977. The secretary IgA system of the gut. Ciba Found. Symp. 46:5-22. 6. Field, M. 1980. Intestinal secretion and its stimulation by enterotoxins. Nobel Symp. 43:46-52. 7. Haskins, W. T., and P. P. Weinstein. 1957. The amine

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10.

11.

12.

13.

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constitutents from the excretory products of Ascaris lumbricoides and Trichinella spiralis larvae. J. Parasitol. 43:28-32. Holmgren, J., S. Lange, and I. Lmnnroth. 1978. Inhibition of chlorpromazine of cyclic-AMP-mediated intestinal secretion. Gastroenterology 75:1103-1109. Johnson, L R., and A. M. Chandler. 1973. RNA and DNA of gastric and duodenal mucosa in antrectomized and gastrin-treated rats. Am. J. Physiol. 224:937-940. Lange, S., H.-A. Hanason, S.-O. Molin, and H. Nygren. 1979. Local cholera immunity in mice: intestinal antitoxin-containing cells and their correlation with protective immunity. Infect. Immun. 23:743-750. Lange, S., and J. Holmgren. 1978. Protective antitoxic cholera immunity in mice: influence of route and number of immunizations and mode of action of protective antibodies. Acta Pathol. Microbiol. Scand. Sect. C 86: 145-152. Lange, S., H. Nygren, A.-M. Svennerholm, and J. Holmgren. 1980. Antitoxic cholera immunity in mice: influence of antigen deposition on antitoxin-containing cells and protective immunity in different parts of the intestine. Infect. Immun. 28:17-23. Lichtenberger, L., L. R. Miller, D. N. Erwin, and L

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14.

15.

16.

17.

18.

19.

R. Johnson. 1973. Effect of pentagastrin on adult rat duodenal cells in culture. Gastroenterology 65:242-251. Ljungstrom, L. 1974. Antibody response to Trichinella spiralis, p. 449-459. In C. W. Kim (ed.), Proceedings of the Third International Conference on Trichinellosis. Intext Educational, New York. Ljungstrom, I. 1980. Studies on the responsiveness of spleen cells to various polyclonal T and B cell activators during Trichinella spiralis infection. Parasite Immunol. 2:111-120. Ljungstrom, I., and G. Huldt. 1977. Effect of experimental trichinosis on unrelated humoral and cell mediated immunity. Acta Pathol. Microbiol. Scand. Sect. C 85:131-141. Svennerholm, A.-M., S. Lange, and J. Holmgren. 1978. Correlation between intestinal synthesis of specific immunoglobulin A and protection against experimental cholera in mice. Infect. Immun. 21:1-6. Symons, L E. A. 1965. Kinetics of the epithelial cells and morphology of villa and crypts in the jejunum of the rat infected by the nematode Nippostrongylus brasiliensis. Gastroenterology 49:158-168. Terry, R. J. 1977. Immunodepression in parasite infection. INSERM Colloq. 72:161-178.